Skip to main content

Advertisement

Log in

miR-124-3p promotes BMSC osteogenesis via suppressing the GSK-3β/β-catenin signaling pathway in diabetic osteoporosis rats

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

The purpose of this study is to investigate miRNAs’ effects, targeting the Wnt signaling pathway, on osteogenic differentiation to provide new targets for diabetic osteoporosis treatments. Twelve male rats were divided into a normal rat group (NOR group) and a model rat group (MOD group). Cluster analysis of differentially expressed miRNAs and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. Primary rat bone marrow mesenchymal stem cells (BMSCs) were divided into a high-glucose group and a low-glucose group, and osteogenic differentiation was induced. Alkaline phosphatase (ALP) staining and Alizarin Red staining were used for pathological analysis of the cells. Western blot analysis was used to measure GSK-3β, β-catenin, p-β-catenin, c-Myc, and CyclinD1 expression. Immunofluorescence (IF) was used to analyze the effect of GSK-3β inhibitor (CHIR99021) on β-catenin and CyclinD1 expressions levels in BMSCs. A total of 428 differentially expressed miRNAs were found between the NOR and MOD groups. KEGG analysis showed that the target genes were mostly enriched in signaling pathways, including PI3K-Akt, focal adhesion, AGE-RAGE, HIF-1, and Wnt. qPCR verification demonstrated that miR-124-3p exhibited the greatest difference in expression level. In BMSCs, miR-124-3p overexpression could reverse the inhibited expression of BMSC osteogenic markers, including Alpl, Bglap, and Runx2, induced by high glucose. Western blot analysis revealed that the transfection of miR-124-3p mimics could further reverse the upregulated p-β-catenin and GSK-3β levels and the downregulated c-Myc and CyclinD1 levels induced by high glucose. IF results revealed that BMSCs treated CHIR99021 under high glucose showed the reduced GSK-3β and increased β-catenin and CyclinD1 expression levels. Our research highlighted miRNAs’ important roles in regulating the Wnt pathway and provided new information for the diagnosis and treatment of diabetic osteoporosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Baron R, Kneissel M (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19:179–192

    Article  CAS  Google Scholar 

  • Bedene A, Mencej Bedrac S, Jese L, Marc J, Vrtacnik P, Prezelj J, Kocjan T, Kranjc T, Ostanek B (2016) MiR-148a the epigenetic regulator of bone homeostasis is increased in plasma of osteoporotic postmenopausal women. Wien Klin Wochenschr 128:519–526

    Article  CAS  Google Scholar 

  • Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, Hankenson KD, MacDougald OA (2005) Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A 102:3324–3329

    Article  CAS  Google Scholar 

  • Bodnar L, Stanczak A, Cierniak S, Smoter M, Cichowicz M, Kozlowski W, Szczylik C, Wieczorek M, Lamparska-Przybysz M (2014) Wnt/beta-catenin pathway as a potential prognostic and predictive marker in patients with advanced ovarian cancer. J Ovarian Res 7:16

    Article  Google Scholar 

  • Chatterjee S, Khunti K, Davies MJ (2017) Type 2 diabetes. Lancet 389:2239–2251

    Article  CAS  Google Scholar 

  • Day TF, Guo X, Garrett-Beal L, Yang Y (2005) Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 8:739–750

    Article  CAS  Google Scholar 

  • Fan C, Jia L, Zheng Y, Jin C, Liu Y, Liu H, Zhou Y (2016) MiR-34a promotes osteogenic differentiation of human adipose-derived stem cells via the RBP2/NOTCH1/CYCLIN D1 coregulatory network. Stem Cell Reports 7:236–248

    Article  CAS  Google Scholar 

  • Feng Q, Zheng S, Zheng J (2018) The emerging role of microRNAs in bone remodeling and its therapeutic implications for osteoporosis. Biosci Rep 38

  • Heilmeier U, Hackl M, Skalicky S, Weilner S, Schroeder F, Vierlinger K, Patsch JM, Baum T, Oberbauer E, Lobach I, Burghardt AJ, Schwartz AV, Grillari J, Link TM (2016) Serum miRNA signatures are indicative of skeletal fractures in postmenopausal women with and without type 2 diabetes and influence osteogenic and adipogenic differentiation of adipose tissue-derived mesenchymal stem cells in vitro. J Bone Miner Res 31:2173–2192

    Article  CAS  Google Scholar 

  • Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C (2005) Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 8:727–738

    Article  CAS  Google Scholar 

  • Hsu MN, Huang KL, Yu FJ, Lai PL, Truong AV, Lin MW, Nguyen NTK, Shen CC, Hwang SM, Chang YH, Hu YC (2020) Coactivation of endogenous Wnt10b and Foxc2 by CRISPR activation enhances BMSC osteogenesis and promotes calvarial bone regeneration. Mol Ther 28:441–451

    Article  CAS  Google Scholar 

  • Hu C, Jia W (2018) Diabetes in China: epidemiology and genetic risk factors and their clinical utility in personalized medication. Diabetes 67:3–11

    Article  CAS  Google Scholar 

  • Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F (2005) Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 132:49–60

    Article  CAS  Google Scholar 

  • Huang W, Yang S, Shao J, Li YP (2007) Signaling and transcriptional regulation in osteoblast commitment and differentiation. Front Biosci 12:3068–3092

    Article  CAS  Google Scholar 

  • Huelsken J, Behrens J (2002) The Wnt signalling pathway. J Cell Sci 115:3977–3978

    Article  CAS  Google Scholar 

  • Kim JH, Liu X, Wang J, Chen X, Zhang H, Kim SH, Cui J, Li R, Zhang W, Kong Y, Zhang J, Shui W, Lamplot J, Rogers MR, Zhao C, Wang N, Rajan P, Tomal J, Statz J, Wu N, Luu HH, Haydon RC, He TC (2013) Wnt signaling in bone formation and its therapeutic potential for bone diseases. Ther Adv Musculoskelet Dis 5:13–31

    Article  CAS  Google Scholar 

  • Li KC, Chang YH, Yeh CL, Hu YC (2016a) Healing of osteoporotic bone defects by baculovirus-engineered bone marrow-derived MSCs expressing MicroRNA sponges. Biomaterials 74:155–166

    Article  CAS  Google Scholar 

  • Li J, Feng Z, Chen L, Wang X, Deng H (2016b) MicroRNA-335-5p inhibits osteoblast apoptosis induced by high glucose. Mol Med Rep 13:4108–4112

    Article  CAS  Google Scholar 

  • Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20:781–810

    Article  CAS  Google Scholar 

  • Mathieu PS, Loboa EG (2012) Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape, mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways. Tissue Eng Part B Rev 18:436–444

    Article  CAS  Google Scholar 

  • Mohsin S, Baniyas MM, AlDarmaki RS, Tekes K, Kalasz H, Adeghate EA (2019) An update on therapies for the treatment of diabetes-induced osteoporosis. Expert Opin Biol Ther 19:937–948

    Article  CAS  Google Scholar 

  • Moriwaki Y, Yamamoto T, Higashino K (1999) Enzymes involved in purine metabolism--a review of histochemical localization and functional implications. Histol Histopathol 14:1321–1340

    CAS  PubMed  Google Scholar 

  • Prins HJ, Braat AK, Gawlitta D, Dhert WJ, Egan DA, Tijssen-Slump E, Yuan H, Coffer PJ, Rozemuller H, Martens AC (2014) In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells. Stem Cell Res 12:428–440

    Article  CAS  Google Scholar 

  • Qian C, Zhu C, Yu W, Jiang X, Zhang F (2015) High-fat diet/low-dose streptozotocin-induced type 2 diabetes in rats impacts osteogenesis and Wnt signaling in bone marrow stromal cells. PLoS One 10:e0136390

    Article  Google Scholar 

  • Rossini M, Gatti D, Adami S (2013) Involvement of WNT/beta-catenin signaling in the treatment of osteoporosis. Calcif Tissue Int 93:121–132

    Article  CAS  Google Scholar 

  • Sato M, Ye W, Sugihara T, Isaka Y (2016) Fracture risk and healthcare resource utilization and costs among osteoporosis patients with type 2 diabetes mellitus and without diabetes mellitus in Japan: retrospective analysis of a hospital claims database. BMC Musculoskelet Disord 17:489

    Article  Google Scholar 

  • Schwartz AV (2017) Efficacy of osteoporosis therapies in diabetic patients. Calcif Tissue Int 100:165–173

    Article  CAS  Google Scholar 

  • Sealand R, Razavi C, Adler RA (2013) Diabetes mellitus and osteoporosis. Curr Diab Rep 13:411–418

    Article  CAS  Google Scholar 

  • Sita-Lumsden A, Dart DA, Waxman J, Bevan CL (2013) Circulating microRNAs as potential new biomarkers for prostate cancer. Br J Cancer 108:1925–1930

    Article  CAS  Google Scholar 

  • Wang L, Gao P, Zhang M, Huang Z, Zhang D, Deng Q, Li Y, Zhao Z, Qin X, Jin D, Zhou M, Tang X, Hu Y, Wang L (2017) Prevalence and ethnic pattern of diabetes and prediabetes in China in 2013. Jama 317:2515–2523

    Article  Google Scholar 

  • Wang L, Zhao X, Wei BY, Liu Y, Ma XY, Wang J, Cao PC, Zhang Y, Yan YB, Lei W, Feng YF (2015) Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway. Biochimie 108:85–93

    Article  CAS  Google Scholar 

  • Wilczynska A, Bushell M (2015) The complexity of miRNA-mediated repression. Cell Death Differ 22:22–33

    Article  CAS  Google Scholar 

  • You L, Pan L, Chen L, Gu W, Chen J (2016) MiR-27a is essential for the shift from osteogenic differentiation to adipogenic differentiation of mesenchymal stem cells in postmenopausal osteoporosis. Cell Physiol Biochem 39:253–265

    Article  CAS  Google Scholar 

  • Zhang J, Tu Q, Bonewald LF, He X, Stein G, Lian J, Chen J (2011) Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1. J Bone Miner Res 26:1953–1963

    Article  CAS  Google Scholar 

  • Zhao R, Li Y, Lin Z, Wan J, Xu C, Zeng Y, Zhu Y (2016) miR-199b-5p modulates BMSC osteogenesis via suppressing GSK-3beta/beta-catenin signaling pathway. Biochem Biophys Res Commun 477:749–754

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the Shenzhen Municipal Science and Technology Bureau (JCYJ20160428180924481) and Sanming Project of Medicine in Shenzhen (SZSM201512043).

Author information

Authors and Affiliations

Authors

Contributions

HL conceived and designed the study, and critically revised the manuscript. ZL performed the experiments, analyzed the data, and drafted the manuscript. HZ, SC, XL, XQ, JL, and DL participated in study design, study implementation, and manuscript revision. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Huilin Li.

Ethics declarations

Conflict of interests

The authors declare that they have no conflict of interests.

Ethics approval

All procedures performed in studies involving animals were in accordance with the ethical standards of Guangdong Province Experimental Animal Center.

Additional information

Editor: Tetsuji Okamoto

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Z., Zhao, H., Chu, S. et al. miR-124-3p promotes BMSC osteogenesis via suppressing the GSK-3β/β-catenin signaling pathway in diabetic osteoporosis rats. In Vitro Cell.Dev.Biol.-Animal 56, 723–734 (2020). https://doi.org/10.1007/s11626-020-00502-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-020-00502-0

Keywords

Navigation